CN108088830B - A method for accurate determination of chitosan oligosaccharide content by resonance Rayleigh scattering - Google Patents
A method for accurate determination of chitosan oligosaccharide content by resonance Rayleigh scattering Download PDFInfo
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- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 title claims abstract description 82
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- 238000001514 detection method Methods 0.000 claims abstract description 14
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
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- 238000005070 sampling Methods 0.000 claims description 5
- 229920000742 Cotton Polymers 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
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- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000784732 Lycaena phlaeas Species 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical group CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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Abstract
本发明涉及一种通过共振瑞利散射法准确测定壳寡糖含量的方法，属于大分子检测领域。本发明利用一定条件下，壳寡糖能与宝石红结合形成离子缔合物，使共振散射强度显著增强，加入十二烷基硫酸钠作为增敏剂提高其灵敏度。通过共振散射强度与壳寡糖在一定的浓度范围内呈线性关系，以此作为壳寡糖的定量基础，建立了测定壳寡糖的共振瑞利散射法。本方法测定受离子浓度和干扰物的影响，在实际样品测定中，需控制离子浓度在0.05mol/L‑0.20mol/L的标准品做定量标准，具有试剂廉价，灵敏度高，重现性好和操作简便等优点。
The invention relates to a method for accurately determining the content of chitosan oligosaccharide by a resonance Rayleigh scattering method, and belongs to the field of macromolecular detection. The invention utilizes that under certain conditions, chitosan oligosaccharide can be combined with ruby red to form an ion association, so that the resonance scattering intensity is significantly enhanced, and sodium lauryl sulfate is added as a sensitizer to improve its sensitivity. Through the linear relationship between resonance scattering intensity and chitosan oligosaccharide in a certain concentration range, as the quantitative basis of chitosan oligosaccharide, a resonance Rayleigh scattering method for the determination of chitosan oligosaccharide was established. The determination of this method is affected by the ion concentration and interfering substances. In the actual sample determination, the standard substance with the ion concentration of 0.05mol/L-0.20mol/L needs to be controlled as the quantitative standard. It has the advantages of cheap reagents, high sensitivity and good reproducibility. and easy operation.
Description
Technical Field
The invention relates to a method for accurately measuring the content of chitosan oligosaccharide by a resonance Rayleigh scattering method, belonging to the field of macromolecular detection.
Background
Chitooligosaccharide (COS), also known as low-oligo-glucose, low-oligo-glucosamine, is a basic amino oligosaccharide formed by 2-glucosamine linked by β -1, 4 glycosidic bond, and contains a part of acetylglucosamine residues. The chitosan oligosaccharide has wide sources, safety, no toxicity, good water solubility, and multiple biological activities of oxidation resistance, bacteriostasis, tumor resistance, immunity enhancement and the like, and has attracted wide attention of medical researchers at home and abroad. Therefore, the accurate quantification of the chitosan oligosaccharide is very important, and the establishment of a simple and efficient method for measuring the content of the chitosan oligosaccharide is of great significance, so that the application of the chitosan oligosaccharide in various fields is further promoted.
The existing common methods for measuring chitosan oligosaccharide still have some defects, such as colorimetric method: the method can accurately judge the average oligochitosan degree through gel permeation chromatography verification. Although the colorimetric method has simple technology, low requirement on equipment and low cost, the accuracy is not high, so that a new analysis method capable of accurately measuring the chitosan oligosaccharide is needed to be developed; chromatography: although the thin layer chromatography can analyze the chitosan oligosaccharide and has low price, the method has long operation time, only can carry out micro-production and cannot carry out quantification, and the thin layer-colorimetric method can be used for routine detection of the chitosan oligosaccharide. However, there is a need for a new technique for large-scale separation and analysis of chitooligosaccharides. At present, the related research for directly measuring the content of the chitosan oligosaccharide by using a simple, convenient, rapid and high-sensitivity resonance Rayleigh scattering method is less at home and abroad, so that the method for measuring the chitosan oligosaccharide by using the resonance Rayleigh scattering method has great research space and significance.
Based on the method, the invention provides a method for accurately measuring the content of the chitosan oligosaccharide by a resonance Rayleigh scattering method.
Disclosure of Invention
In order to overcome the technical defects that the content of chitosan oligosaccharide in a finished product is difficult to quantify and the operation technology is complicated in the prior art, the invention provides the method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method. The resonance Rayleigh scattering method for measuring the chitosan oligosaccharide is established by taking the linear relation between the resonance scattering intensity and the chitosan oligosaccharide in a certain concentration range as the quantitative basis of the chitosan oligosaccharide. The method is influenced by ion concentration and interferents, and in the actual sample measurement, a standard substance with the ion concentration of 0.05-0.20 mol/L needs to be controlled to serve as a quantitative standard, so that the method has the advantages of low reagent price, high sensitivity, good reproducibility, simplicity and convenience in operation and the like.
A method for accurately measuring the content of chitosan oligosaccharide by a resonance Rayleigh scattering method comprises the following steps:
1) drawing a standard curve of delta I and chitosan oligosaccharide with different concentrations
Adding 1.0mL of B-R buffer solution, a chitosan oligosaccharide standard solution with a certain concentration gradient, 1.0mL of sensitizer with the concentration of 0.086mol/L and 1.0mL of sensitizer with the concentration of 1.0 multiplied by 10 into a 10mL colorimetric tube-4Adding distilled water to desired volume, shaking thoroughly, placing at room temperature for 10min, and placing on F-2500 type fluorescence spectrophotometer at lambdaex＝λemSynchronous scanning is carried out, and the resonance scattering intensity I of each detection system containing the chitosan oligosaccharide at the position of lambda-470 nm is recorded, wherein the resonance scattering intensity I of the system of a reagent blank at the position of lambda-470 nm is recorded0And calculating the value of Delta I ═ I-I0Establishing a standard curve C of the delta I and the concentration of the chitosan oligosaccharide;
2) preparation of 10. mu.g/mL sample working solution: removing the capsule shell of the chitosan oligosaccharide capsule, weighing 0.04g of the chitosan oligosaccharide capsule into a 100mL volumetric flask, dissolving the chitosan oligosaccharide capsule in 0.5mol/L glacial acetic acid, and fixing the volume to obtain a sample stock solution. Filtering the stock solution with absorbent cotton in a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifuge, taking 2.5mL of supernatant in a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
3) Determination of the content of chitosan oligosaccharide in the sample: sampling 1mL of working solution, measuring according to the detection method in the step 1), measuring the scattering value I at 470nm, and calculating delta I-I0Substituting the delta I into a linear regression equation to obtain the content of the chitosan oligosaccharide in the sample, and simultaneously performing a standard addition recovery test.
The method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method is characterized in that the B-R buffer solution is prepared by 0.04mol/L mixed acid and 0.2mol/L NaOH solution according to different proportions.
The method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method is characterized in that the mixed acid is prepared from orthophosphoric acid, glacial acetic acid and boric acid according to different proportions.
The method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method has the advantages that the pH value of the B-R buffer solution is 4.9.
In the method for accurately determining the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method, the sensitizer is one of Tween 20, Tween 80, OP emulsifier, sodium dodecyl sulfate or sodium dodecyl sulfate
In the method for accurately determining the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method, the sensitizer is sodium dodecyl sulfate.
In the method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method, the adding sequence of the solution in the step 1 is that the ruby red solution is firstly added, then the sodium dodecyl sulfate solution is added, the RB buffer solution is added again, and finally the chitosan oligosaccharide solution is added.
The method for accurately measuring the content of the chitosan oligosaccharide by the resonance Rayleigh scattering method has the concentration range of the chitosan oligosaccharide of 0.5 mu g/mL-2.5 mu g/mL.
Sample pretreatment: removing the capsule shell of the chitosan oligosaccharide capsule, weighing 0.04g of the chitosan oligosaccharide capsule into a 100mL volumetric flask, dissolving the chitosan oligosaccharide capsule in 0.5mol/L glacial acetic acid, and fixing the volume to obtain a sample stock solution. Filtering the stock solution with absorbent cotton in a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifuge, taking 2.5mL of supernatant in a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
Sampling 1mL of working solution, measuring according to an experimental method, measuring the absorbance value I at 470nm, and calculating delta I-I0Substituting the delta I into a linear regression equation to obtain the content of the chitosan oligosaccharide in the sample, and simultaneously performing a standard addition recovery test.
Test example:
1. resonance Rayleigh scattering spectrum of chitosan oligosaccharide-ruby red-sodium dodecyl sulfate system
FIG. 1 is a resonance scattering spectrum of the system. As can be seen from fig. 1, the scattering values of COS, SDS and ruby red themselves are low, but the scattering value is significantly increased when COS is combined with all three of ruby red and SDS, and there is a scattering peak at 470 nm. The blank group of the experiment has a lower scattering value, and the experimental group increases the scattering value of the system along with the increase of the concentration of COS, so that a better linear relation exists.
Effect of the pH of the BR buffer solution on the Scattering value of the System
To a 10mL cuvette, 2.0mL of a buffer solution B-R having a pH of 3, 4, 5, 6, 1.0mL of a 10. mu.g/mL chitosan oligosaccharide solution, and 2.0mL of a 1.0X 10 solution were added-4And (3) fixing the volume of the ruby red solution of mol/L to a scale with triple distilled water, fully shaking up, oscillating for 90s, and standing at normal temperature for 30 min. On a F-2500 type spectrofluorometer with a lambdaex＝λemAnd carrying out synchronous scanning to obtain a resonance Rayleigh scattering spectrum.
As a result, as shown in fig. 2 and fig. 3, the acidity of the buffer solution has a significant influence on the reaction system, and the scattering value is large at around pH 5.0, and the final optimum acidity condition is determined at pH 4.9 by a refinement experiment of acidity.
3. Effect of different buffers on the Scattering value of the System
The influence of 3 buffer solutions, namely B-R buffer solution, citric acid-sodium citrate buffer solution and glycine-hydrochloric acid buffer solution, on the scattering value of the system is respectively examined. The scattering values of the respective buffer solutions were obtained by changing the buffer solution added according to the experimental method.
As shown in FIGS. 2, 4 and 5, the system has the highest sensitivity and the largest scattering value in the B-R buffer solution, so the best buffer solution is selected as the B-R buffer solution.
4. Effect of buffer addition on Scattering values
The buffer solution provides an appropriate acidity binding environment for the experimental system, the addition amount of the buffer solution has certain influence on the formation of the ionic associate, other experimental conditions are not changed, the addition amount of the B-R buffer solution with the pH of 4.9 is changed to 0.5, 1.0, 1.5, 2.0, 2.5 and 3mL, and the scattering value is measured.
As a result, as shown in FIG. 6, the B-R buffer solution added in an amount of 1.5mL and 2.5mL showed peaks, and the Δ I was larger at 1.5mL, so that the B-R buffer solution was added in an amount of 1.5 mL.
5. Effect of Gem Red addition on Scattering values
Geranium red as a dye probe is combined with target substance chitosan oligosaccharide, the addition amount of the Geranium red directly influences the formation of ion association, other experimental conditions are not changed, and the concentration is changed to be 1.0 multiplied by 10-4The amount of ruby red added was 0.5, 1.0, 1.5, 2.0, 2.5, 3mL in mol/L, and the scattering value was measured.
The results are shown in fig. 7, where the amount of ruby red added has a significant effect on the reaction system, and it can be seen that as the ruby red dye solution is added, Δ I rises first and then falls, and peaks at an addition of 2 mL. Therefore, the adding amount of the ruby red is 2 mL.
6. Influence of sensitizer addition on Scattering values
The influence of 5 surfactants including Tween 20, Tween 80, OP emulsifier, Sodium Dodecyl Sulfate (SDS) and sodium dodecyl sulfate (SLS) on the scattering value of the system is examined. According to the experimental method, other experimental conditions are not changed, and finally 1mL of surfactant is added, one group is kept stand at normal temperature, the other group is heated in a water bath at 80 ℃ for 10min and then kept stand to normal temperature, and the scattering value is measured.
The results are shown in fig. 8, when sodium dodecyl sulfate is added, the scattering value of the system is obviously enhanced, and the results show that the adding amount of the sodium dodecyl sulfate has certain influence on the system, so the Sodium Dodecyl Sulfate (SDS) flushing solution is selected as the sensitizer for the experiment.
7. Effect of sodium dodecyl sulfate addition on Scattering values
Sodium dodecyl sulfate is an anionic surfactant, the critical micelle concentration of the surfactant is 0.0086mol/L, and when the Critical Micelle Concentration (CMC) is reached, surfactant molecules are mutually aggregated by van der Waals force to form micelles with oleophilic groups inward and hydrophilic groups outward, so that the solubility of ion associations in a micelle solution is remarkably increased, and the resonance scattering strength is remarkably increased by the generated micelle solubilization. The scattering values were determined by changing the amount of sodium lauryl sulfate added at a concentration of 0.086mol/L to 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3mL without changing other experimental conditions.
The result is shown in fig. 9, and the result shows that the addition of sodium dodecyl sulfate has a certain influence on the system, the overall trend is increased first and then decreased, the scattering value is maximum when the addition is 1mL, and then slightly decreased, the scattering value is larger when the addition is 1.0mL, and the stability of the system is good, so that the optimal addition of 0.086mol/L sodium dodecyl sulfate is 1.0mL, namely, the corresponding concentration of sodium dodecyl sulfate is 0.0086 mol/L.
8. Effect of the order of reagent addition on the Scattering value of the System
Under experimental conditions, the influence of 12 different adding sequences of anionic dyes ruby red, B-R buffer solution, COS and SDS on the sensitivity of the system is examined. The results are shown in Table 1, FIG. 10, and the optimal sequence of addition is "Gem Red + SDS + RB buffer + COS", where the system has the highest scattering values, the highest sensitivity and the best reproducibility.
TABLE 1 Effect of the order of addition
9. Influence of reaction temperature on the scattering value of the System
Under experimental conditions, the influence of different temperatures of 20-100 ℃ on the sensitivity of the system is examined, the result is shown in FIG. 11, and the result shows that the temperature has influence on the scattering value of the system. The scattering value is in the trend of rising first, then falling and then rising along with the rising of the temperature, the maximum scattering value is reached at 20 ℃, and the repeatability is better at the reaction temperature, so the optimal temperature of the system is 20 ℃.
10. Effect of the settling time on the Scattering value of the System
And under the better condition, the stable time of the system is inspected, the stable time is inspected for 3h, and the scattering value is measured at intervals. The result shows that the system can reach stability and the scattering value reaches the maximum within 10min, and the stable scattering value can be kept unchanged within 2 h. Therefore, the stabilization time was selected to be 2 h.
11. Effect of ion intensity on the Scattering value of the System
And (3) examining the influence of the ionic strength on the scattering value of the system by using NaCl (0.005-0.3 mol/L). As a result, as shown in FIG. 12, the ion concentration was in the range of 0.05mol/L to 0.2mol/L, the scattering value tended to be stable, and the system was relatively stable.
12. Effect of coexisting materials on the Scattering value of the System
The interference of 6 coexisting substances on the system is examined, and the concentration of the chitosan oligosaccharide in the system is 1 mug/mL. The allowable amounts of the respective coexisting materials within. + -. 5% relative errors are shown in Table 2. The interference of common copper, calcium, manganese and the like is small, and the allowable amount is large.
TABLE 2 Effect of coexisting materials
Compared with the prior art, the invention has the following technical advantages:
1) good linearity and low detection limit: under the best experimental condition, the corresponding delta I of the chitosan oligosaccharide with different concentrations is measured according to the experimental method, a standard curve is drawn, and the result shows that the chitosan oligosaccharide has good linear relation with the delta I within the range of 0.5 mu g/mL-2.5 mu g/mL, the linear range is wide, and the detection limit is 0.4708 mu g/mL.
2) The method is influenced by ion concentration and interferents, and in the actual sample measurement, a standard substance with the ion concentration of 0.05-0.20 mol/L needs to be controlled to serve as a quantitative standard, so that the method has the advantages of low reagent price, high sensitivity, good reproducibility, simplicity and convenience in operation and the like.
Drawings
FIG. 1 resonance Rayleigh scattering spectra of the chitooligosaccharide-ruby red-sodium lauryl sulfate system.
FIG. 2 is a graph showing the effect of pH of BR buffer on the scattering value of the system.
FIG. 3 shows a detailed pH map of BR buffer.
FIG. 4 is a graph showing the effect of pH of glycine-hydrochloric acid buffer on the scattering value of the system.
FIG. 5 is a graph showing the effect of pH of a citric acid-sodium citrate buffer on the scattering value of a system.
FIG. 6 is a graph showing the effect of the amount of buffer added on the scattering value of the system.
FIG. 7 is a graph showing the effect of ruby red addition on system scatter values.
FIG. 8 is a graph showing the effect of different sensitizers on the scattering value of the system.
FIG. 9 is a graph showing the effect of SDS addition on the scattering value of the system.
FIG. 10 is a graph showing the effect of different reagent addition sequences on the scattering value of the system.
FIG. 11 is a graph of the effect of temperature on the scattering value of a system.
FIG. 12 graph of the effect of ionic strength on scattering values of a system
Detailed Description
The invention is further described below by means of specific examples, but said invention is not in any way restricted to the scope of the invention as claimed.
Examples
A method for accurately measuring the content of chitosan oligosaccharide by a resonance Rayleigh scattering method comprises the following steps:
1) drawing a standard curve of delta I and chitosan oligosaccharide with different concentrations
Adding 1.0mL of B-R buffer solution with pH of 4.9, a chitosan oligosaccharide standard solution with a concentration gradient, 1.0mL of sodium dodecyl sulfate solution with concentration of 0.086mol/L and 1.0mL of sodium dodecyl sulfate solution with concentration of 1.0 multiplied by 10 into a 10mL colorimetric tube-4Adding distilled water to desired volume, shaking thoroughly, placing at room temperature for 10min, and placing on F-2500 type fluorescence spectrophotometer at lambdaex＝λemSynchronous scanning is carried out, and the resonance scattering intensity I of each detection system containing the chitosan oligosaccharide at the position of lambda-470 nm is recorded, wherein the resonance scattering intensity I of the system of a reagent blank at the position of lambda-470 nm is recorded0And calculating the value of Delta I ═ I-I0And establishing a standard curve C of the delta I and the concentration of the chitosan oligosaccharide.
2) Preparation of 10. mu.g/mL sample working solution: removing the capsule shell of the chitosan oligosaccharide capsule, weighing 0.04g of the chitosan oligosaccharide capsule into a 100mL volumetric flask, dissolving the chitosan oligosaccharide capsule in 0.5mol/L glacial acetic acid, and fixing the volume to obtain a sample stock solution. Filtering the stock solution with absorbent cotton in a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifuge, taking 2.5mL of supernatant in a 100mL volumetric flask, and performing constant volume to obtain a sample working solution with the concentration of 10 mug/mL.
And (3) drawing a sample curve by using the sample working solution according to an experimental method, and comparing the sample curve with a chitosan oligosaccharide standard curve to obtain a linear regression equation of the sample, wherein the linear regression equation is that delta I is 33.31c +151.41, and the correlation coefficient is 0.9901.
3) Determination of the content of chitosan oligosaccharide in the sample: sampling 1mL of working solution, measuring according to the detection method in the step 1), measuring the scattering value I at 470nm, and calculating delta I-I0Substituting the delta I into a linear regression equation to obtain the content of the chitosan oligosaccharide in the sample, and simultaneously performing a standard addition recovery test.
Sampling 1mL of working solution, measuring according to an experimental method, measuring a scattering value I at 470nm, and calculating delta I-I0Substituting the delta I into a linear regression equation to obtain the content of the chitosan oligosaccharide in the sample, and simultaneously performing a standard addition recovery test. The results were: the content of the chitosan oligosaccharide in the olygoxate chitosan oligosaccharide capsule is 1174mg/g, and the RSD is 2.081%. The recovery rates are shown in Table 3.
TABLE 3 results of sample analysis
In the experiment, 2.0mL of colorimetric tubes with a concentration of 1.0X 10 were sequentially added-4Ruby red in mol/L, sodium lauryl sulfate in 1.0mL at a concentration of 0.086mol/L, B-R buffer solution in 1.5mL at pH 4.9, and 10. mu.g in 3.0mLAnd (3) diluting the chitosan oligosaccharide solution to a certain volume by using distilled water, fully shaking up, and standing for 10min at room temperature. On a F-2500 type fluorescence spectrophotometer, as lambdaex＝λemSynchronous scanning is carried out, and the determination is completed within 2 h. Recording the resonance scattering intensity I of the chitosan oligosaccharide-containing system at λ 470nm, wherein the resonance scattering intensity I of the reagent blank system at λ 470nm0And calculating the value of Delta I ═ I-I0. The chitosan oligosaccharide has good linear relation with Delta I within the concentration range of 0.5 mu g/mL-2.5 mu g/mL, the linear range is wide, and the detection limit is 0.4708 mu g/mL. The method is influenced by ion concentration and interferents, and in the actual sample measurement, a standard substance with the ion concentration of 0.05-0.20 mol/L needs to be controlled to serve as a quantitative standard, so that the method has the advantages of low reagent price, high sensitivity, good reproducibility, simplicity and convenience in operation and the like.
Linear range and detection limit
Under the best experimental conditions, the corresponding delta I of chitosan oligosaccharide with different concentrations is determined according to an experimental method, a standard curve is drawn, the result shows that the chitosan oligosaccharide has a good linear relation with the delta I in the concentration range of 0.5 mu g/mL-2.5 mu g/mL, the linear regression equation is that the delta I is 33.31c +151.41, the correlation coefficient is 0.9901, the method linear range is wide, and the detection limit is 0.4708 mu g/mL.
Claims (1)
1. A method for accurately measuring the content of chitosan oligosaccharide by a resonance Rayleigh scattering method is characterized by comprising the following steps:
1) drawing a standard curve of delta I and chitosan oligosaccharide with different concentrations to a 10mL colorimetric tube, adding 1.0mL of B-R buffer solution, a chitosan oligosaccharide standard solution with a certain concentration gradient, 1.0mL of sensitizer with the concentration of 0.086mol/L and 1.0mL of sensitizer with the concentration of 1.0 multiplied by 10-4Adding distilled water to desired volume, shaking thoroughly, placing at room temperature for 10min, and placing on F-2500 type fluorescence spectrophotometer at lambdaex＝λemSynchronous scanning is carried out, the resonance scattering intensity I of each detection system containing the chitosan oligosaccharide at the position of lambda-470 nm is recorded, the resonance scattering intensity I0 of the system at the position of lambda-470 nm of a reagent blank is recorded, and delta I-I is calculated0Establishing a standard of Delta I and chitosan oligosaccharide concentrationA quasi-curve C;
2) preparation of 10. mu.g/mL sample working solution: removing the capsule shell of the chitosan oligosaccharide capsule, weighing 0.04g of chitosan oligosaccharide capsule in a 100mL volumetric flask, dissolving with 0.5mol/L glacial acetic acid, diluting to a constant volume to obtain a sample stock solution, filtering the stock solution with absorbent cotton through a funnel, centrifuging the filtrate for 20min at 6000r/min by a centrifugal machine, taking 2.5mL of supernatant in the 100mL volumetric flask, diluting to a constant volume to obtain a sample working solution with the concentration of 10 mug/mL;
3) determination of the content of chitosan oligosaccharide in the sample: sampling 1mL of working solution, measuring according to the detection method in the step 1), measuring the scattering value I at 470nm, and calculating delta I-I0Substituting the delta I into a linear regression equation to obtain the content of the chitosan oligosaccharide in the sample, and simultaneously performing a standard addition recovery test;
the B-R buffer solution is prepared by 0.04mol/L mixed acid and 0.2mol/L NaOH solution according to different proportions; the mixed acid is prepared from orthophosphoric acid, glacial acetic acid and boric acid according to different proportions; the pH value of the B-R buffer solution is 4.9; the sensitizer is sodium dodecyl sulfate; the adding sequence of the solution in the step 1 is that firstly, a ruby red solution is added, secondly, a sodium dodecyl sulfate solution is added, thirdly, an RB buffer solution is added, and finally, a chitosan oligosaccharide solution is added; the concentration range of the chitosan oligosaccharide is 0.5 mu g/mL-2.5 mu g/mL.
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